PROJECT SUMMARY Our long-term objective is to characterize the microenvironments that influence B lymphocyte development during altered bone homeostasis. B lymphocytes are required for the production of antibodies that are crucial for a robust adaptive immune response. Studies from many groups have determined the stages of maturation, the molecular mechanisms of B cell antigen receptor gene rearrangements and generation of receptor diversity, and mechanisms of self-tolerance in conventional B cells (also called ?B2? cells). The microenvironments in the bone marrow (BM) that support B2 progenitors and mature B2 cells have been described (2-7), and it appears that development of robust antibody responses from B2 cells is influenced by the relatively low oxygen levels in the germinal centers of the spleen and lymph nodes (11). However, relatively little is known about the mechanisms by which oxygen levels regulate B cell development in the bone marrow. We aim to fill the scientific knowledge gap on the microenvironmental niches that influence the maintenance and function of adult B cell subsets. In the course of our studies to examine the effects of altered bone homeostasis on immune cell development, we discovered that B cell development was severely impaired in mice in which a hypoxia response pathway gene, von-Hippel Lindau (Vhl), is conditionally deleted in osteocytes (12). We hypothesize that Vhl-deficiency in osteocytes results in structural and molecular changes in the vascular architecture in the BM microenvironments, which may alter oxygen tension to levels that support aberrant B2 development. We further hypothesize that molecular changes in the osteocytes results in a microenvironment that insufficiently produces cytokines to appropriately support B cell maturation. In support of these hypotheses, our preliminary analyses suggest that deletion of Vhl in osteocytes results in increased permeability in the vasculature. Our project objective is to further characterize the osteocyte-regulated microenvironmental influences on the development and maintenance of B lymphocytes because of Vhl deficiency. We aim to utilize high-resolution intravital (live) and ex vivo organ imaging and transplantation strategies to characterize the Vhl-deficient bone marrow microenvironment in order to identify changes that may influence B cell development, such as oxygen tension, blood vessel type and structure, the presence and location of stromal cells or hematopoietic cells, and changes in bone morphology. In parallel, we will also use cytokine immunoassays and co-culture methods to identify the molecular changes in the VhlcKO BM stromal cell niches. Finally, we aim to analyze the niches longitudinally over time, in order to determine how the distinct physical properties in the bone change and affects the progression of B cell developmental defects in VhlcKO bones as a function of time. This information could be applied to future studies of the effects of irradiation, myeloablative conditioning, or bone-building drugs on bone marrow niches and immune cell development.